Many studies have been undergoing about RNA tumor virus, particularly Moloney-Murine Leukemia Virus (M-MLV), human HIV, or Avian Myeloblastosis Virus (AMV) originated reverse transcriptases and accordingly their functions and properties have been disclosed. Reverse transcriptase has been used for many molecular biological methods such as cDNA library construction, reverse transcription and polymerase chain reaction (PCR), etc, owing to its unique characteristics favoring the synthesis of complementary DNA (cDNA) by using RNA as a template.
Three prototypes of retrovirus reverse transcriptase have been mainly studied. Moloney-Murine Leukemia Virus originated reverse transcriptase contains 78 kDa single subunit having RNA dependent DNA polymerase and RNase H activity. The said enzyme is cloned and expressed in E. coli as an authentic active form. HIV originated reverse transcriptase is hetero-dimer of p66 and p51 subunits. P51 subunit is generated by proteolytic cleavage of p66 subunit. P66 subunit contains both RNA dependent DNA polymerase and RNase H domains, but p51 subunit contains only DNA polymerase domain. Active HIV originated p66/p51 reverse transcriptase is cloned and expressed in many expression hosts including E. coli. In HIV p66/p51 hetero-dimer, 51 kDa subunit is catalytically inactive and 66 kDa subunit shows both DNA polymerase activity and RNase H activity. In the meantime, Avian Sarcoma-Leukosis Virus (ASLV) reverse transcriptases such as Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus reverse transcriptase, Avian Erythroblastosis Virus (AEV) helper virus MCAV reverse transcriptase, Avian Myelocytomatosis Virus MC29 helper virus MCAV reverse transcriptase, Avian Reticuloendotheliosis Virus (REV-T) helper virus REV-A reverse transcriptase, Avian Sarcoma Virus UR2 helper virus UR2AV reverse transcriptase, Avian Sarcoma Virus Y73 helper virus YAV reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, and Myeloblastosis Associated Virus (MAV) reverse transcriptase are hetero-dimer of two subunits of alpha (approximately 62 kDa) and beta (approximately 94 kDa). Alpha subunit is generated by proteolytic cleavage of beta subunit. ASLV reverse transcriptase can exist in two catalytically active structures of alpha/beta and beta/beta. From the sediment analysis, it was confirmed that the said alpha/beta and beta/beta structures are dimmers and the alpha herein exists in the equilibrium state between monomer and dimer. ASLV alpha/beta or beta/beta reverse transcriptase is the only retrovirus reverse transcriptase informed up to date to have three different activities such as DNA polymerase activity, RNase H activity, and DNA endonuclease (integrase) activity. The alpha structure has neither integrase domain nor its activity.
The conversion of mRNA to cDNA by reverse transcriptase mediated reverse transcription is very important in the studies on gene expression. However, it is not preferred in many ways to use untransformed reverse transcriptase as a mediator for reverse transcription. Reverse transcriptase happens to degrade RNA template by RNase H activity before the first strand reaction begins or when it is completed. There is also a chance of error in the first strand of cDNA by mis-priming of mRNA template. In fact, during cDNA synthesis, HIV reverse transcriptase makes errors as many as 1 nucleotide error per 3,000-6,000 nucleotides, while AMV reverse transcriptase makes 1 nucleotide error per 10,000 nucleotides.
Another factor that affects the effect of reverse transcriptase is whether RNA can form the secondary structure or not. Such secondary structure can be formed when RNA molecular has enough complementarity to generate double-stranded RNA. In general, the formation of RNA secondary structure can be reduced by increasing the temperature of RNA molecule containing solution. So, it is preferred to perform reverse transcription of RNA at higher than 37° C. The reverse transcriptase of the present invention loses its activity when the culture is performed at much higher temperature than 37° C. (for example, at 50° C.)
A variety of methods to process thermostable reverse transcriptase are informed in the prior art, which include the method using thermostable DNA polymerase having reverse transcriptase activity, the method increasing reverse transcriptase activity by inducing mutation in thermostable DNA polymerase, the method inducing mutation in thermo-unstable reverse transcriptase, the method using Mn2+ instead of Mg2+ in the presence of Taq/Tth DNA polymerase, and the method using such additive as trehalose along with thermo-unstable reverse transcriptase.
To increase fidelity of polymerization of DNA or RNA template, those people skilled in the art have been using various enzyme compositions and methods. For example, Shevelev et al. provide a review article about 3′-5′ exonuclease (Shevelev et al., Nature Rev. Mol. Cell. Biol. 3:364 (2002)). Perrino et al. use the upsilon subunit of E. coli DNA polymerase III to increase fidelity of calf thymus DNA polymerase alpha (Perrino et al., PNAS USA, 86:3085 (1989)). Bakhanashvili explains proofreading activity of p53 protein (Bakhanashvili, Eur. J. Biochem. 268:2047 (2001)), while Huang et al. describe the use of p53 to increase fidelity of DNA replication (Huang et al., Oncogene, 17:261 (1998)). US Patent Publication No. 2003/0198944A1 and U.S. Pat. No. 6,518,19 describe the enzyme mixture containing one or more reverse transcriptases (each reverse transcriptase has different transcription termination site) and if necessary containing one or more DNA polymerases additionally. US Patent Publication No. 2002/0119465A1 describes the composition containing mutant thermostable DNA polymerase and mutant reverse transcriptase (for example, mutant Taq DNA polymerase and mutant MMLV-RT). U.S. Pat. No. 6,485,917B1, US Patent Publication No. 2003/0077762, and European Patent Publication No. EP1132470 describe the method of cDNA synthesis in the presence of α-type DNA polymerase having 3′-5′ exonuclease activity and the enzyme having reverse transcriptase activity.
When RNase H activity of reverse transcriptase is eliminated, the problem of RNA degradation of RNA template can be excluded, and further reverse transcription efficiency can be improved. However, such reverse transcriptase (‘RNase H−’ type) cannot solve the problems of mis-priming and the generation of mRNA secondary structure. The conventional reverse transcriptase has low thermostability, and thus reverse transcription is only induced at comparatively low temperature. Accordingly, reverse transcription product cannot be efficiently obtained by the interruption of RNA secondary structure formed generally at as high temperature as at least 65° C. Such limitation is a major barrier not only for the synthesis of cDNA from the full-length RNA but also for various biochemical experiments requiring reverse transcription such as RNA detection and profiling. Therefore, it is highly required to develop a novel reverse transcriptase having stable reverse transcription activity even at high temperature.